[unreadable] Gamma-secretase-mediated intramembrane proteolysis of the amyloid precursor protein, Notch, and select transmembrane transcription factors represents a novel mechanism of signal transduction. Aberrant proteolysis of the amyloid precursor protein, which gives rise to amyloid beta-peptides, is a central event in the pathogenesis of Alzheimer's disease. It is now believed that gamma-secretase is a multimeric, high-molecular-weight complex that contains at least four membrane proteins: presenilin, nicastrin, APH-1, and PEN-2. The precise roles of these four proteins are not known, nor is how the unusual gamma-secretase assembles to specifically cleave peptide bonds of select membrane proteins within a hydrophobic environment. It is also not clear how gamma-secretase substrates, which share no sequence homology, are selected and recruited to the gamma-secretase complex for cleavage. We hypothesize that assembly and enzymatic activity of the gamma-secretase complex is governed by intermolecular interactions among the four proteins, while substrate specificity is controlled by the nicastrin ectodomain, both of which are likely aided by additional unknown molecules. This model will be tested in the following four hypothesis-driven specific aims. First, in vitro experimental systems for studying gamma-secretase and its individual components will be established. Second, the assembly and proteolytic activity of the gamma-secretase complex will be examined by defining the direct binding partners and the docking sites of nicastrin, presenilin, APH-1, and PEN-2; and by determining the size, stoichiometry, and enzymatic activity of the complex of the four proteins. Third, whether nicastrin is a molecular sensor for gamma-secretase substrate specificity will be tested by examining whether nicastrin selects and recruits gamma-secretase substrates, and by identifying potential ligands for the ectodomain of nicastrin. Fourth, additional proteins that associate with nicastrin, APH-1, and PEN-2 will be identified and their effects on gamma-secretase activity will be characterized. These studies should provide crucial insights into the molecular nature and biochemical mechanism of the gamma-secretase complex, and should reveal new targets and avenues for designing better therapeutic strategies for Alzheimer's disease and related disorders. [unreadable] [unreadable]